A detailed understanding of the processes that control axon growth and guidance is essential for understanding the development of the nervous system and for engineering successful regrowth and reconnection of severed neurons following spinal cord injury. Many of the signaling pathways responsible for enhancing or inhibiting growth and for attractive and repulsive guidance have been identified. However, the vast majority of our knowledge of the mechanisms of axon motility come from studies of growth cones navigating on flat, stiff substrates. There is considerable evidence that cell motility in complex, pliable, three dimensional (3D) extracellular environments can be very different than on a coated glass coverslip. The goal of this exploratory research proposal is to develop the tools necessary to probe the cytoskeletal dynamics of growth cones in 3D collagen matrices and to initiate studies of the cytoskeletal response to extracellular guidance cues in 3D. Our specific aims are to: (1) Measure growth cone cytoskeletal dynamics in 3D collagen gels. (2) Quantify the effect of gradients of guidance factors on growth cone cytoskeletal dynamics in 3D. The development of these techniques to investigate the cytoskeletal dynamics of guidance in 3D gels will lay the groundwork for addressing fundamental questions about the biophysical and biochemical mechanisms that underlie axon motility, and will lay the groundwork for the development of controlled manipulations of the environment that impact axon motility in predictable ways. We will be in a position to generate models of inhibitory cues encountered during development or in the glial scar, analyze the mechanisms by which those cues affect axon motility, and then systematically manipulate the environment and the signal transduction pathways to validate hypothesis about the mechanisms of axon guidance or to test interventions to control axon growth. [unreadable] [unreadable]